Junqi Ling

Stem Cell Regulatory Gene Expression in Human Adult Dental Pulp and Periodontal Ligament Cells Undergoing Odontogenic/Osteogenic Differentiation

INTRODUCTION During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. METHODS In this study, we investigated the differential expression of 84 stem cell-related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. RESULTS Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. CONCLUSIONS This study has generated an overview of stem cell-related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-beta/BMP, and cadherin signaling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration.

Biofilm Formation Capability of Enterococcus faecalis Cells in Starvation Phase and Its Susceptibility to Sodium Hypochlorite

INTRODUCTION Enterococcus faecalis is commonly associated with persistent periapical infections. The physiologic state of the cells in the canal is probably closest to the starvation state. However, the biofilm formation capability of starved E. faecalis cells on human dentin and the susceptibility of the biofilm to 5.25% sodium hypochlorite remain poorly understood. METHODS E. faecalis American Type Culture Collection (ATCC) 29212 in different growth phases were incubated on human dentin and polystyrene blocks. Scanning electron microscopy and biofilm assay were used to investigate the biofilm formation capability of these cells. The susceptibility of the biofilm to 5.25% NaClO was also determined by the plate count method. RESULTS Scanning electron microscopy and biofilm assay showed that starved cells were able to form biofilm on dentin with reduced efficiency as compared with the cells in the exponential phase and stationary phase (p < 0.05). Biofilm grown on dentin harbored more cells than polystyrene (p < 0.05). Biofilms of starved cells were more resistant to 5.25% NaClO than those of stationary cells (p < 0.05), and the impact of 5.25%NaClO on them decreased as the biofilm matured. CONCLUSION E. faecalis cells in the starvation phase could develop biofilm on human dentin, which is responsive to 5.25% NaClO. It may contribute to the predominant role of E. faecalis involved in persistent periapical infections.

A microcomputed tomographic study of canal isthmuses in the mesial root of mandibular first molars in a Chinese population.

Untreated isthmuses can be a cause of endodontic treatment failure. We investigated the anatomic features of the isthmus in the mesial root of mandibular first molars using microcomputed tomography scans. Thirty-six extracted mandibular first molars were collected from the Chinese population and divided into three age groups as follows: 20 to 39 years (group A), 40 to 59 years (group B), and > or = 60 years (group C). Each tooth was scanned and reconstructed, and then the prevalence and type of isthmus were recorded. The percentage of sections showing isthmuses for groups A, B, and C were 50%, 41%, and 24%, respectively. The chi-square test indicated a significant correlation of the distribution of isthmuses with age (p < 0.001). The ratio of partial isthmus to complete isthmus for group C (17.1:1) was significantly higher than group A (5.9:1) and group B (7.0:1) (p < 0.001). By understanding the configuration and location of isthmus, a more efficient endodontic microsurgery can be guaranteed.

Effects of Irrigation Solutions on Dentin Wettability and Roughness

INTRODUCTION Irrigation solutions are used to disinfect the root canal, flush out debris, and dissolve pulp tissue. It is hypothesized that irrigation solutions might influence the physicochemical properties of human root canal dentin including the wettability and roughness. METHODS The roots of 20 human permanent anterior teeth were sectioned longitudinally into dentin slices and randomly divided into 4 groups with different treatments as follows: (1) 17% ethylenediaminetetraacetic acid (EDTA) for 10 minutes, (2) 5.25% NaOCl for 10 minutes, (3) 3% H2O2 for 10 minutes, and (4) distilled water as control. Wettability was evaluated by contact angle measurement. Atomic force microscopy was used to measure the surface roughness (Ra). RESULTS Water contact angles significantly decreased from 54.35 degrees (control) to 22.04 and 46.35 degrees on the dentin treated by 5.25% NaOCl and 3% H2O2, respectively, with the wettability increasing accordingly (both P < .05). The roughness of dentin surfaces (Ra) was increased from 85.39 (control) to 121.90 and 140.49 nm by 5.25% NaOCl and 17% EDTA, respectively (both P < .05). CONCLUSIONS These findings indicated that all the irrigation solutions could alter the physicochemical characteristics of dentin surfaces and might influence bacterial adhesion.

Expression Pattern of Oct-4, Sox2, and c-Myc in the Primary Culture of Human Dental Pulp Derived Cells

Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage (P < .05), and then decreased along the number of passages afterwards (P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions.

The expression of stromal cell-derived factor 1 (SDF-1) in inflamed human dental pulp.

Although dental pulp progenitor/stem cells (DPSCs) are indispensable for repair after pulpal injury, the mechanisms regulating their recruitment and activation remain unknown. To address this issue, we evaluated whether DPSCs in inflamed dental pulp had an upregulation of the chemokine system, a system of proteins known to regulate cellular responses to inflammation. Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine subfamily and its receptor CXC chemokine receptor 4 (CXCR4), were evaluated in inflamed dental pulps obtained from extracted human teeth that showed spontaneous pain and/or lingering pain in response to cold and/or heat stimulus and compared with control levels found in normal dental pulps obtained from healthy noncarious third molars. Using immunohistochemistry and real-time reverse-transcription polymerase chain reaction, the results indicated that in inflamed pulps the SDF-1/CXCR4 axis was mostly distributed in inflammatory cells and microvascular endothelial cells rather than in normal pulps. SDF-1 messenger RNA expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. These findings suggest that SDF-1 plays an important role in the process of pulpal inflammation via the recruitment of CXCR4-expressing inflammatory cells, and the SDF-1/CXCR4 axis may be involved in the recruitment of dental pulp stem cells at the injury site.

Antimicrobial and DNA-binding activities of the peptide fragments of human lactoferrin and histatin 5 against Streptococcus mutans

OBJECTIVE To investigate the killing effect of two salivary antimicrobial peptides, hLF1-11 and P-113, and identify the antibacterial mechanism of the peptides. METHODS The antimicrobial activities of hLF1-11 and P-113 against oral Streptococci strains were determined using the broth microdilution method. The effects of hLF1-11 and P-113 on the bacterial plasma membrane were visualized by scanning electron microscopy. Cell membrane permeability was monitored using the intracellular dye calcein. The subcellular localization of hLF1-11 and P-113 in bacteria was measured by fluorescence light microscopy. An electrophoretic mobility shift assay (EMSA) was performed to evaluate the DNA binding capabilities of hLF1-11, P-113 and MUC7 12-mer. RESULTS Both hLF1-11 and P-113 exerted potent bactericidal activities against all selected oral Streptococci. Streptococcus mutans UA 159 was the most susceptible of the oral bacterial species tested to the antimicrobial effects of the three peptides. The cell membranes of bacteria treated with hLF1-11 or P-113 were still intact after 30 min. hLF1-11 and P-113 could penetrate the bacterial cell membranes and accumulate in the cytoplasm in S. mutans. Both hLF1-11 and P-113 showed DNA binding affinity. CONCLUSIONS Together, our results demonstrate that hLF1-11 and P-113 display antibacterial activity against dental cavity-inducing S. mutans through an intracellular mechanism that could involve DNA binding. Thus, these peptides might be attractive and valuable candidates for development into effective antimicrobial therapies to combat dental caries.

An In Vitro Study on the Effects of Nisin on the Antibacterial Activities of 18 Antibiotics against Enterococcus faecalis

Enterococcus faecalis rank among the leading causes of nosocomial infections worldwide and possesses both intrinsic and acquired resistance to a variety of antibiotics. Development of new antibiotics is limited, and pathogens continually generate new antibiotic resistance. Many researchers aim to identify strategies to effectively kill this drug-resistant pathogen. Here, we evaluated the effect of the antimicrobial peptide nisin on the antibacterial activities of 18 antibiotics against E. faecalis. The MIC and MBC results showed that the antibacterial activities of 18 antibiotics against E. faecalis OG1RF, ATCC 29212, and strain E were significantly improved in the presence of 200 U/ml nisin. Statistically significant differences were observed between the results with and without 200 U/ml nisin at the same concentrations of penicillin or chloramphenicol (p<0.05). The checkerboard assay showed that the combination of nisin and penicillin or chloramphenicol had a synergetic effect against the three tested E. faecalis strains. The transmission electron microscope images showed that E. faecalis was not obviously destroyed by penicillin or chloramphenicol alone but was severely disrupted by either antibiotic in combination with nisin. Furthermore, assessing biofilms by a confocal laser scanning microscope showed that penicillin, ciprofloxacin, and chloramphenicol all showed stronger antibiofilm actions in combination with nisin than when these antibiotics were administered alone. Therefore, nisin can significantly improve the antibacterial and antibiofilm activities of many antibiotics, and certain antibiotics in combination with nisin have considerable potential for use as inhibitors of this drug-resistant pathogen.

Side Population Increase after Simulated Transient Ischemia in Human Dental Pulp Cell

INTRODUCTION Dental pulp is often exposed to ischemia in case of injury or inflammation because of narrow vascular openings at the apex and poor blood circulation in dental pulp tissue. Resident stem cell populations are thought to contribute to the postischemic regeneration process. The aim of this study was to investigate the influence of simulated ischemia (serum deprivation and hypoxia) on side population (SP) stem cells of human dental pulp cells in order to provide a better understanding of the postischemic tissue repair and regeneration process. METHODS The proliferation of dental pulp cells (DPCs) after exposure to ischemic culture conditions (2% O2, 2% serum) for 24 hours and 48 hours was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The SP fraction was detected by Hoechst 33342 fluorescence flow cytometry, and the expression of SP marker ABCG2 was investigated by immunofluorescence. ABCG2 and OCT4 messenger RNA levels before and after transient ischemia were determined by real-time polymerase chain reaction. RESULTS Proliferation rate of DPCs was lower in 24- and 48-hour ischemic groups than control from day 5 to day 7. SP proportion was significantly higher 24 and 48 hours after simulated ischemic treatment, and immunofluorescence staining of ABCG2 also verified the increasing trend of side population. ABCG2 and OCT4 messenger RNA levels increased more than three folds in 48 hours ischemic group compared with control group. CONCLUSIONS Side population in dental pulp cells increase notably after transient simulated ischemic culture, suggesting that SP may participate in post-ischemic repair and regeneration process of dental pulp.

Regulation of the Stromal Cell–derived Factor-1α–CXCR4 Axis in Human Dental Pulp Cells

INTRODUCTION Although the presence of the stromal cell-derived factor (SDF)-1alpha-CXCR4 axis has been reported in dental pulp tissue, little has been known about the underlying regulation of this axis in dental pulp stem cells (DPSCs). The purpose of this study was to investigate whether inflammation or hypoxia can regulate this axis in cultured human dental pulp cells (DPCs). METHODS Primary cultures of DPCs were stimulated by various concentrations of lipopolysaccharide (LPS) for 48 hours, and the production of SDF-1alpha or CXCR4 was assessed through the enzyme-linked immunosorbent assay and Western blotting, respectively. Additionally, DPCs were incubated in a hypoxic condition (1% O(2)) for 24 hours, and the cell proliferation ability was detected by methylthiazol tetrazolum assay. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to observe messenger RNA level changes of hypoxia inducible factor-1alpha(HIF-alpha), SDF-1alpha, and CXCR4. The effects of hypoxia on cell migration ability were further confirmed by transmigration assay. RESULTS All concentrations of LPS inhibited SDF-1alpha production except that 1 microg/mL LPS increased the expression of CXCR4. Hypoxia promoted the proliferation of DPCs in a 24-hour culture period. Quantitative RT-PCR showed that messenger RNA levels of HIF-alpha and CXCR4 increased, whereas SDF-1alpha decreased in hypoxic DPCs. Transmigration assay indicated that hypoxia increased the migration ability of DPCs. CONCLUSIONS These results suggested that inflammation and hypoxia might play an important role in regulating the SDF-1alpha-CXCR4 axis, which further recruits DPSCs to participate in reparative dentinogenesis.